Identification of potential sites for tryptophan oxidation in recombinant antibodies using tert-butylhydroperoxide and quantitative LC-MS

Amino acid oxidation is known to affect the structure, activity, and rate of degradation of proteins. Methionine oxidation is one of the several chemical degradation pathways for recombinant antibodies. In this study, we have identified for the first time a solvent accessible tryptophan residue (Trp...

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Bibliographic Details
Published in:	PloS one Vol. 6; no. 3; p. e17708
Main Authors:	Hensel, Miriam, Steurer, Rebecca, Fichtl, Juergen, Elger, Carsten, Wedekind, Frank, Petzold, Andreas, Schlothauer, Tilman, Molhoj, Michael, Reusch, Dietmar, Bulau, Patrick
Format:	Journal Article
Language:	English
Published:	United States Public Library of Science 03-03-2011 Public Library of Science (PLoS)
Subjects:	Accessibility Amino Acid Sequence Amino acids Antibodies Antibodies - chemistry Antibodies - metabolism Binding Biodegradation Biology Chains Chemical degradation Chemistry Chromatography Chromatography, Liquid Degradation ErbB-2 protein Evaluation High temperature Identification Immunoglobulin G Immunoglobulins Light chains Mass Spectrometry Medicine Methionine Methionine - metabolism Molecular Sequence Data Oxidation Oxidation-Reduction - drug effects Oxidation-reduction reactions Peptides Proteins Proteolysis Real time Recombinant Proteins - chemistry Recombinant Proteins - metabolism Reference materials Reference Standards Retention Solvents Surface Plasmon Resonance t-Butylhydroperoxide Temperature Temperature effects tert-Butylhydroperoxide - pharmacology Tryptophan Tryptophan - metabolism Ultraviolet Rays Gaza Strip Germany
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Summary:	Amino acid oxidation is known to affect the structure, activity, and rate of degradation of proteins. Methionine oxidation is one of the several chemical degradation pathways for recombinant antibodies. In this study, we have identified for the first time a solvent accessible tryptophan residue (Trp-32) in the complementary-determining region (CDR) of a recombinant IgG1 antibody susceptible to oxidation under real-time storage and elevated temperature conditions. The degree of light chain Trp-32 oxidation was found to be higher than the oxidation level of the conserved heavy chain Met-429 and the heavy chain Met-107 of the recombinant IgG1 antibody HER2, which have already been identified as being solvent accessible and sensitive to chemical oxidation. In order to reduce the time for simultaneous identification and functional evaluation of potential methionine and tryptophan oxidation sites, a test system employing tert-butylhydroperoxide (TBHP) and quantitative LC-MS was developed. The optimized oxidizing conditions allowed us to specifically oxidize the solvent accessible methionine and tryptophan residues that displayed significant oxidation in the real-time stability and elevated temperature study. The achieved degree of tryptophan oxidation was adequate to identify the functional consequence of the tryptophan oxidation by binding studies. In summary, the here presented approach of employing TBHP as oxidizing reagent combined with quantitative LC-MS and binding studies greatly facilitates the efficient identification and functional evaluation of methionine and tryptophan oxidation sites in the CDR of recombinant antibodies.
Bibliography:	ObjectType-Article-2 SourceType-Scholarly Journals-1 ObjectType-Feature-1 content type line 23 ObjectType-Article-1 ObjectType-Feature-2 Conceived and designed the experiments: PB MH. Performed the experiments: MH RS JF CE FW AP TS DR. Analyzed the data: PB MH RS MM. Contributed reagents/materials/analysis tools: PB DR. Wrote the paper: PB MM.
ISSN:	1932-6203 1932-6203
DOI:	10.1371/journal.pone.0017708